HPIV1 Human parainfluenza virus type 1 (HPIV1) is a significant cause of severe respiratory tract disease in infants and young children for which a vaccine is needed. The complete genomic sequence of HPIV1 strain Washington/20993/1964 (Wash/64) was recently determined, and wild type recombinant HPIV1 (rHPIV1) was recovered from an HPIV1 antigenomic cDNA. In the present study, we sought to attenuate HPIV1 by the ?importation? of one or more known attenuating point mutations from several heterologous paramyxoviruses into homologous sites in HPIV1. The introduced mutations were derived from three attenuated paramyxoviruses: (i) HPIV3cp45, a live-attenuated HPIV3 vaccine candidate containing multiple attenuating mutations, (ii) the respiratory syncytial virus (RSV) cpts530 virus with an attenuating mutation in the L polymerase protein, and (iii) a murine PIV1 (MPIV1) attenuated by a mutation in the accessory C protein. Although each of the imported mutations had previously been shown to be attenuating in their native background, not all conferred an attenuation phenotype in rHPIV1. However, rHPIV1 mutants bearing a single imported mutation in C, any of three different mutations in L, or a pair of mutations in F exhibited a 100-fold or greater reduction in replication in the upper or lower respiratory tract of hamsters. Both temperature sensitive (ts) (mutations in the L and F proteins) and non-ts (the mutation in the C protein) attenuating mutations were identified. rHPIV1 mutants containing a combination of two or three mutations in L were generated that were more attenuated than viruses bearing the individual mutations, showing that the systematic accretion of mutations can yield progressive increases in attenuation. Hamsters immunized with any of five rHPIV1 mutants bearing one or two mutations developed neutralizing antibodies and were resistant to challenge with wild type HPIV1. Thus, importation of attenuating mutations from heterologous viruses is an effective means for rapidly identifying mutations that attenuate HPIV1 and for generating live-attenuated HPIV1 vaccine candidates. The paramyxovirus large polymerase protein (L) is highly conserved at the amino acid level between human parainfluenza virus type 1 and type 3 (HPIV1 and HPIV3). The Y942H and L992F temperature sensitive (ts) and attenuating amino acid substitution mutations, previously identified in the L polymerase of the HPIV3-cp45 vaccine candidate, were introduced into homologous positions of the L polymerase of recombinant HPIV1 (rHPIV1). In rHPIV1, the Y942H mutation specified the ts phenotype in vitro and the attenuation (att) phenotype in hamsters whereas the L992F mutation specified neither phenotype. Each of these two mutations in both HPIV3-cp45 and rHPIV1 was generated by a single nucleotide substitution and, therefore, had the potential to readily revert to a codon specifying the wild type amino acid residue. To generate rHPIV1 ts viruses with different levels of attenuation and with enhanced genetic stability, HPIV1 recombinant cDNAs were molecularly engineered to specify each of the other 18 amino acids at position 942 and 17 at position 992 of the L protein. Thirteen rHPIV1 codon substitution mutants with alternative amino acid substitutions at position 942 and 10 with substitutions at position 992 were viable. At position 942, rHPIV1 mutants with a similar level of temperature sensitivity and attenuation as the rHPIV1-Y942H mutant were identified, several of which require three nucleotide substitutions in codon 942 to generate a wild type Tyr codon. One such mutant, rHPIV1-Y942A, exhibited increased genetic and phenotypic stability upon serial passage in vitro at successively elevated restrictive temperatures. At position 992, two recombinants, rHPIV1-L992V and rHPIV1-L992C, were obtained that, in contrast to rHPIV1-L992F, possessed the ts and att phenotypes. These findings identify codon substitution mutations that specify increased genetic stability and/or increased attenuation, properties that are highly desirable for mutations present in a live attenuated HPIV1 vaccine. HMPV The growth properties and antigenic relatedness of the CAN98-75 (CAN75) and the CAN97-83 (CAN83) HMPV strains, which represent the two distinct HMPV genetic lineages and exhibit 5% and 63% amino acid divergence in the F and G proteins, respectively, were investigated in vitro and in rodents and non-human primates. Both strains replicated to high titers (>= 6.0 log10) in the upper respiratory tract of hamsters and to moderate titers (>= 3.6 log10) in the lower respiratory tract. The two lineages exhibited 48% antigenic relatedness based on reciprocal cross-neutralization assay using post-infection hamster sera, and infection with each strain provided a high level of resistance to reinfection with the homologous or heterologous strain. Hamsters immunized with a recombinant human parainfluenza virus type 1 (rHPIV1) expressing the fusion F protein of the CAN83 strain (rHPIV1-F83) developed a serum antibody response that efficiently neutralized virus from both lineages and were protected from challenge with either HMPV strain. This indicates that the HMPV F protein is a major antigenic determinant that mediates extensive cross-lineage neutralization and protection. Both HMPV strains replicated to low titers in the upper and lower respiratory tract of rhesus macaques but induced high levels of serum HMPV-neutralizing antibodies effective against both lineages. The level of HMPV replication in chimpanzees was moderately higher, and infected animals developed mild colds. HMPV replicated the most efficiently in the respiratory tract of African green monkeys, and the infected animals developed a high level of HMPV serum neutralizing antibodies (1:500 to 1:1000) effective against both lineages. Reciprocal cross-neutralization assays using post-infection sera from all three primate species indicated that CAN75 and CAN83 are 64%-99% related antigenically. HMPV infected chimpanzees and African green monkeys were highly protected from challenge with the heterologous HMPV strain. Taken together, the results in hamsters and nonhuman primates support the conclusion that the two HMPV genetic lineages are highly related antigenically and are not distinct antigenic subtypes or subgroups as defined by reciprocal cross neutralization in vitro.